The neuronal ceroid lipofuscinoses or Batten disease (BD) is one of a broad class of severe neurodegenerative
diseases that are known as lysosomal storage disorders (LSDs). BD is characterized by the intracellular
accumulation of storage material in neural tissues and progressive loss of neurological functions and vision that
primarily affect children. CLN2 is a specific type of BD that results from mutations in the TPP1 gene causing an
insufficiency or complete lack of a soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Last year, the first
FDA-approved treatment, intraventricular infusions of a recombinant form of human TPP1, was shown to provide
symptomatic improvements in pediatric patients. Unfortunately, this invasive procedure carries a high risk of
adverse effects. The less invasive systemic administration provides no therapeutic benefits, because the blood
brain barrier (BBB) severely restricts transport of macromolecules - including TPP1- to the brain. To circumvent
this problem, we propose using extracellular vesicles (EVs) released by macrophages as biocompatible
nanocarriers for systemic delivery of TPP1. Comprised of cellular membranes with multiple adhesive proteins
on their surface, EVs are known to specialize in cell-cell communications facilitating transport of proteins and
genetic material to target cells.
Our research has previously demonstrated that therapeutic proteins, including TPP1, can be efficiently
incorporated into EVs without losing their biological activity. In particular, TPP1 was loaded into EVs using two
methods: (i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA), or
(ii) loading therapeutic protein TPP1 into naive empty EVs. The resulting EVs carrier ensemble was shown to
readily migrate into the brains of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice upon systemic
administration. Importantly, multiple lines of evidence for therapeutic efficacy were observed in LINCL mice,
including significant neuroprotection and improved life span. Noteworthy, we developed different methods for
EVs isolation, purification, characterization, and storage in sufficient quantities for therapeutic application.
In the current proposal, we will utilize two EV-based formulations of TPP1 to demonstrate the proof of concept
using a BD mouse model, LINCL mice. Planned studies include: (SA1) elucidation the nature of selective
fingerprinting of macrophage-derived EVs, and mechanism of EVs interactions and TPP1 facilitated uptake in
cells of neurovascular unit; (SA2) evaluation of brain bioavailability for EV-TPP1 with MRI and optical imaging in
vivo, and (SA3) validation the therapeutic potential of this novel drug delivery system by measuring its
neuroprotective and anti-inflammatory effects in LINCL mice. The proposed research addresses a critical
problem in the effective delivery of therapeutic proteins to the central nervous system (CNS), and will provide
fundamental insights into, how EVs communicate with target brain cells, and selectively deliver their cargo.
